Aav-vectors for use in gene therapy of choroideremia

ABSTRACT

The present invention relates to gene therapy for treatment or prevention of choroideremia.

FIELD OF THE INVENTION

The present invention relates to gene therapy for treatment orprevention of choroideremia.

BACKGROUND OF THE INVENTION

Choroideremia is a rare X-linked progressive degeneration of thechoroid, retinal pigment epithelium and photoreceptors of the eye. Thetypical natural history in afflicted males is onset of nightblindncssduring teenage years, and then progressive loss of peripheral visionduring the 20's and 30's leading to complete blindness in the 40's.Female carriers have mild symptoms most notably nightblindncss but mayoccasionally have a more severe phenotype.

The disease is caused by mutations in the REP1 gene, (Rab escort protein1), which is located on the X chromosome 21q region. In most cells inthe body, the REP2 protein, which is 75% homologous to REP1, compensatesfor the REP1 deficiency. In the eye, however, for reasons that are notyet clear, REP2 is unable to compensate for the REP1 deficiency. Hencein the eye, REP polypeptide activity is insufficient to maintain normalprenylation of the target proteins (Rab GTPases) leading to cellulardysfunction and ultimate death, primarily affecting the outer retina andchoroid.

There is no treatment for choroideremia, and there is a lack of modelsto assess therapeutic strategies. There is a need for provision of sucha therapy.

SUMMARY OF THE INVENTION

The present invention relates to a vector which can be used for genetherapy of choroideremia, and methods of preventing or treating thisdisease using the vector. The invention also relates to the use of thevector in methods of preventing or treating choroideremia.

The vector of the invention is a viral vector, specifically based on thegenome of adeno-associated virus (AAV). The vector comprises a sequencewhich encodes REP1 or a variant thereof, thus allowing for theexpression of REP1 function in a target cell. The methods and uses ofthe invention specifically involve the administration of the vector to apatient by direct retinal, subretinal or intravitreal injection to treator prevent choroideremia.

Accordingly, the invention provides a vector, which comprises anadeno-associated virus (AAV) genome or a derivative thereof and apolynucleotide sequence encoding REP1 or a variant thereof The inventionfurther provides a method of treating or preventing choroideremia in apatient in need thereof, comprising administering a therapeuticallyeffective amount of a vector according to any one of the precedingclaims to said patient by direct retinal, subretinal or intravitrealinjection, and thereby treating or preventing choroideremia in saidpatient. The invention additionally provides a vector of the inventionfor use in a method of treating or preventing choroideremia byadministering said vector to a patient by direct retinal, subretinal orintravitreal injection.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows that the AAV.REP1 (AAV-CAG-REP1) vector can transduce humanfibroblasts isolated from a patient with choroideremia (Chm)efficiently. The relative levels of expression of human REP1 protein(hREP1) are compared by Western blot, allowing quantification ofAAV2.REP1 vector activity by comparing the amount of hREP1 in differentconcentrations of cell lysate. With regard to the labelling, CAG is theChicken beta Actin with CMV enhancer promoter sequence—interchangeablyreferred to as ‘CBA’ in various publications and parts of this document.

Western blots are shown in the left panels for REP1 (top panel) andalpha-tubulin (bottom panel) as a loading control. Lane 1: 40 μg celllysate from control wildtype (WT) fibroblasts. Lane 2: 40 μg cell lysatefrom Chm fibroblasts. Lanes 3-6: 40, 20, 10 and 5 μg cell lysate fromChm fibroblasts transduced with AAV2.REP1 vector. Lane 7: human REP1recombinant protein. Since the 5 μg lysate hREP1 band has a similardensity to 40 μg of the WT fibroblast lysate, the level of hREP1achieved with the AAV2.REP1 vector can achieve at least 8 times (40/5)the normal wild type levels under these conditions.

As a positive control for the promoter and other non-REP1 sequences inthis assay, results from a control AAV vector expressing greenfluorescent protein (GFP) in place of REP1 (AAV-CAG-GFP) are also shown.

Western blots are shown in the right panels for GFP (top panel) andalpha-tubulin (bottom panel) as a loading control. Lane 1: 40 μg celllysate from wildtype (WT) fibroblasts. Lane 2: 40 μg cell lysate fromChm fibroblasts transduced with AAV2.GFP vector (AAV-CAG-REP1). The highlevels of GFP shown confirm the efficiency of this vector expressioncassette in transducing human cells that are deficient of REP1 activity,as would be the case in patients with choroideremia.

FIG. 2 shows an assessment of prenylation activity in WT humanfibroblasts (first column on left—light grey), Chm fibroblasts (secondcolumn—dark grey), AAV-CAG-GFP vector-transduced Chm fibroblasts (thirdcolumn—white, negative control) and AAV-CAG-REP1 transduced Chmfibroblasts (fourth column—white). The y axis shows the amount ofradioactively labelled substrate [3H] GGPP substrate transferred inpmol, which is a measure of prenylation, the function of REP1. Columnsshow error bars as standard deviations (n=4 for each column). Levels of[3H]-GGPP were measured in 10 mg of protein extract. The cyan columnfourth from left confirms that the function of prenylation is alsorestored to wild type levels and beyond, following transduction with theAAV.REP1 vector. This confirms that the REP1 protein detected by Westernblot in FIG. 1 has the predicted function.

FIG. 3 shows that the AAV vector has the correct tropism for cells ofthe outer retina (photoreceptors and choroid) following subretinalinjection in a mouse model. The right panel shows appropriate expressionof a green fluorescent protein (GFP) marker (arrows) in thephotoreceptors of the outer nuclear layer (ONL) and retinal pigmentepithelium (RPE) following subretinal injection of theAAV2.CBA.GFP.WPRE.BGH vector in the mouse eye. Left panel showsgreyscale of same image. This confirms that the AAV2.CBA.WPRE.BGHregulatory sequences are capable of highly efficient transgeneexpression in the retinal cells that need to be targeted in patientswith choroideremia.

FIG. 4 shows that the AAV.REP1 vector does not adversely affect outerretinal function at high doses in the mouse retina. The results of atoxicity study with measurement of an electroretinogram (ERG) six monthsafter subretinal injection of 2×1 micro litre of either high (n=5) orlow (n=4) doses of AAV.REP1 vector into the mouse subretinal space areshown. Low dose=1×10¹¹ and high dose=1×10¹² genome particles (gp) per ml(the starting dose for human clinical trials is 1×10¹¹ gp per ml). TheAAV.GFP vector has an identical expression cassette and is also dilutedto the same dose prior to injection to act as a control. The Y axisshows the ERG trace at increasing flash intensities ranging fromscotopic (dark adapted) rod responses above to bright to photopicresponses below (which would also include cone photoreceptors). Thetraces at all points show similar ERG amplitudes following both high andlow dose AAV.REP1 exposure. In the high dose group the equivalent GFPamplitudes are slightly reduced, in keeping with the known marginaleffect on retinal function of GFP at high levels. This GFP effect alsoacts as a positive control to confirm the sensitivity of this test.

DESCRIPTION OF SEQUENCES

SEQ ID NO: 1 is a DNA sequence for the AAV2 genome.

SEQ ID NO: 2 is a DNA sequence encoding the human Rep-1 protein,transcript variant 1.

SEQ ID NO: 3 is an amino acid sequence for the human Rep-1 protein,transcript variant 1.

SEQ ID NO: 4 is a DNA sequence encoding the human Rep-1 protein,transcript variant 1, which includes a portion of the 5′UTR.

SEQ ID NO: 5 is a DNA sequence for the woodchuck hepatitispostregulatory element (WPRE).

SEQ ID NO: 6 is a DNA sequence for a chicken beta actin (CBA) promoter.

SEQ ID NO: 7 is a DNA sequence for a polyadenylation site from BovineGrowth Hormone (bGH polyA).

SEQ ID NO: 8 is a DNA sequence for a 5′inverted terminal repeat (ITR) ofAAV2.

SEQ ID NO: 9 is a DNA sequence for a 31TR of AAV2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a therapy for choroideremia. This isbased on a gene therapy approach to the disease utilising a geneticconstruct to deliver a transgene to restore REP1 function. The geneticconstruct is a vector based on an adeno-associated virus (AAV) genomewhich comprises a polynucleotide sequence encoding REP1 or a variantthereof. This polynucleotide sequence is also referred to herein as the“transgene”. The present inventors established a model for evaluatingstrategies for treatment of choroideremia and surprisingly demonstrateuse of a vector of the invention to target the cellular dysfunctionunderlying the disease.

Vector AAV Genome

The vector of the invention comprises firstly an adeno-associated virus(AAV) genome or a derivative thereof.

An AAV genome is a polynucleotide sequence which encodes functionsneeded for production of an AAV viral particle. These functions includethose operating in the replication and packaging cycle for AAV in a hostcell, including encapsidation of the AAV genome into an AAV viralparticle. Naturally occurring AAV viruses are replication-deficient andrely on the provision of helper functions in trans for completion of areplication and packaging cycle. Accordingly, the AAV genome of thevector of the invention is typically replication-deficient.

The AAV genome may be in single-stranded form, either positive ornegative-sense, or alternatively in double-stranded form. The use of adouble-stranded form allows bypass of the DNA replication step in thetarget cell and so can accelerate transgene expression.

The AAV genome may be from any naturally derived serotype or isolate orclade of AAV. Thus, the AAV genome may be the full genome of a naturallyoccurring AAV virus. As is known to the skilled person, AAV virusesoccurring in nature may be classified according to various biologicalsystems.

Commonly, AAV viruses are referred to in terms of their serotype. Aserotype corresponds to a variant subspecies of AAV which owing to itsprofile of expression of capsid surface antigens has a distinctivereactivity which can be used to distinguish it from other variantsubspecies. Typically, a virus having a particular AAV serotype does notefficiently cross-react with neutralising antibodies specific for anyother AAV serotype. AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11, also recombinant serotypes,such as Rec2 and Rec3, recently identified from primate brain.

A preferred serotype of AAV for use in the invention is AAV2. An AAV2genome may have the sequence of SEQ ID NO: 1. Other serotypes ofparticular interest for use in the invention include AAV4, AAV5 and AAV8which efficiently transduce tissue in the eye, such as the retinalpigmented epithelium. The serotype of AAV which is used can be an AAVserotype which is not AAV4. Reviews of AAV serotypes may be found inChoi et al (Curr Gene Ther. 2005; 5(3); 299-310) and Wu et al (MolecularTherapy. 2006; 14(3), 316-327). The sequences of AAV genomes or ofelements of AAV genomes including ITR sequences, rep or cap genes foruse in the invention may be derived from the following accession numbersfor AAV whole genome sequences: Adeno-associated virus 1 NC_002077,AF063497; Adeno-associated virus 2 NC_001401; Adeno-associated virus 3NC_001729; Adeno-associated virus 3B NC_001863; Adeno-associated virus 4NC_001829; Adeno-associated virus 5 Y18065, AF085716; Adeno-associatedvirus 6 NC_001862; Avian AAV ATCC VR-865 AY186198, AY629583, NC_004828;Avian AAV strain DA-1 NC_006263, AY629583; Bovine AAV NC_005889,AY388617.

AAV viruses may also be referred to in terms of clades or clones. Thisrefers to the phylogenetic relationship of naturally derived AAVviruses, and typically to a phylogenetic group of AAV viruses which canbe traced back to a common ancestor, and includes all descendantsthereof. Additionally, AAV viruses may be referred to in terms of aspecific isolate, i.e. a genetic isolate of a specific AAV virus foundin nature. The term genetic isolate describes a population of AAVviruses which has undergone limited genetic mixing with other naturallyoccurring AAV viruses, thereby defining a recognisably distinctpopulation at a genetic level.

Examples of clades and isolates of AAV that may be used in the inventioninclude: Clade

A: AAV1 NC_002077, AF063497, AAV6 NC_001862, Hu. 48 AY530611, Hu 43AY530606, Hu 44 AY530607, Hu 46 AY530609

Clade B: Hu. 19 AY530584, Hu. 20 AY530586, Hu 23 AY530589, Hu22AY530588, Hu24 AY530590, Hu21 AY530587, Hu27 AY530592, Hu28 AY530593, Hu29 AY530594, Hu63 AY530624, Hu64 AY530625, Hu13 AY530578, Hu56 AY530618,Hu57 AY530619, Hu49 AY530612, Hu58 AY530620, Hu34 AY530598, Hu35AY530599, AAV2 NC 001401, Hu45 AY530608, Hu47 AY530610, Hu51 AY530613,Hu52 AY530614, Hu T41 AY695378, Hu. S17 AY695376, Hu T88 AY695375, HuT71 AY695374, Hu T70 AY695373, Hu T40 AY695372, Hu. T32 AY695371, Hu.T17 AY695370, Hu LG15 AY695377,

Clade C: Hu9 AY530629, HulO AY530576, Hull AY530577, Hu53 AY530615, Hu55AY530617, Hu54 AY530616, Hu7 AY530628, Hu18 AY530583, Hul5 AY530580,Hu16 AY530581, Hu25 AY530591, Hu60 AY530622, Ch5 AY243021, Hu3 AY530595,Hu1 AY530575, Hu4 AY530602 Hu2, AY530585, Hu61 AY530623

Clade D: Rh62 AY530573, Rh48 AY530561, Rh54 AY530567, Rh55 AY530568, Cy2AY243020, AAV7 AF513851, Rh35 AY243000, Rh37 AY242998, Rh36 AY242999,Cy6 AY243016, Cy4 AY243018, Cy3 AY243019, Cy5 AY243017, Rh13 AY243013

Clade E: Rh38 AY530558, Hu66 AY530626, Hu42 AY530605, Hu67 AY530627,Hu40 AY530603, Hu41 AY530604, Hu37 AY530600, Rh40 AY530559, Rh2AY243007, Bb1 AY243023, Bb2 AY243022, Rh10 AY243015, Hu17 AY530582, Hu6AY530621, Rh25 AY530557, Pi2 AY530554, Pil AY530553, Pi3 AY530555, Rh57AY530569, Rh50 AY530563, Rh49 AY530562, Hu39 AY530601, Rh58 AY530570,Rh61 AY530572, Rh52 AY530565, Rh53 AY530566, Rh51 AY530564, Rh64AY530574, Rh43 AY530560, AAV8 AF513852, Rh8 AY242997, Rh1 AY530556

Clade F: Hu14 (AAV9) AY530579, Hu31 AY530596, Hu32 AY530597, ClonalIsolate AAV5 Y18065, AF085716, AAV 3 NC_001729, AAV 3B NC_001863, AAV4NC_001829, Rh34 AY243001, Rh33 AY243002, Rh32 AY243003/

The skilled person can select an appropriate serotype, clade, clone orisolate of AAV for use in the present invention on the basis of theircommon general knowledge. For instance, the AAV5 capsid has been shownto transduce primate cone photoreceptors efficiently as evidenced by thesuccessful correction of an inherited color vision defect (Mancuso etal., Nature 2009, 461:784-7).

It should be understood however that the invention also encompasses useof an AAV genome of other serotypes that may not yet have beenidentified or characterised. The AAV serotype determines the tissuespecificity of infection (or tropism) of an AAV virus. Accordingly,preferred AAV serotypes for use in AAV viruses administered to patientsin accordance with the invention are those which have natural tropismfor or a high efficiency of infection of target cells within thedegenerating retina in choroideremia. Thus, preferred AAV serotypes foruse in AAV viruses administered to patients arc ones which infect cellsof the neurosensory retina and retinal pigment epithelium.

Typically, the AAV genome of a naturally derived serotype or isolate orclade of AAV comprises at least one inverted terminal repeat sequence(ITR). An ITR sequence acts in cis to provide a functional origin ofreplication, and allows for integration and excision of the vector fromthe genome of a cell. In preferred embodiments, one or more ITRsequences flank the polynucleotide sequence encoding Rep-1 or a variantthereof. Preferred ITR sequences are those of AAV2, including those ofSEQ ID NOs 8 and 9 and variants thereof The AAV genome typically alsocomprises packaging genes, such as rep and/or cap genes which encodepackaging functions for an AAV viral particle. The rep gene encodes oneor more of the proteins Rep78, Rep68, Rep52 and Rep40 or variantsthereof The cap gene encodes one or more capsid proteins such as VP1,VP2 and VP3 or variants thereof These proteins make up the capsid of anAAV viral particle. Capsid variants are discussed below.

A promoter will be operably linked to each of the packaging genes.Specific examples of such promoters include the p5, p19 and p40promoters (Laughlin et al., 1979, PNAS, 76:5567-5571). For example, thep5 and p19 promoters are generally used to express the rep gene, whilethe p40 promoter is generally used to express the cap gene.

As discussed above, the AAV genome used in the vector of the inventionmay therefore be the full genome of a naturally occurring AAV virus. Forexample, a vector comprising a full AAV genome may be used to prepareAAV virus in vitro. However, while such a vector may in principle beadministered to patients, this will be done rarely in practice.Preferably the AAV genome will be derivatised for the purpose ofadministration to patients. Such derivatisation is standard in the artand the present invention encompasses the use of any known derivative ofan AAV genome, and derivatives which could be generated by applyingtechniques known in the art. Derivatisation of the AAV genome and of theAAV capsid are reviewed in Coura and Nardi (Virology Journal, 2007,4:99), and in Choi et al and Wu et al, referenced above.

Derivatives of an AAV genome include any truncated or modified forms ofan AAV genome which allow for expression of a Rep-1 transgene from avector of the invention in vivo. Typically, it is possible to truncatethe AAV genome significantly to include minimal viral sequence yetretain the above function. This is preferred for safety reasons toreduce the risk of recombination of the vector with wild-type virus, andalso to avoid triggering a cellular immune response by the presence ofviral gene proteins in the target cell.

Typically, a derivative will include at least one inverted terminalrepeat sequence (ITR), preferably more than one ITR, such as two ITRs ormore. One or more of the ITRs may be derived from AAV genomes havingdifferent serotypes, or may be a chimeric or mutant ITR. A preferredmutant ITR is one having a deletion of a trs (terminal resolution site).This deletion allows for continued replication of the genome to generatea single-stranded genome which contains both coding and complementarysequences i.e. a self-complementary AAV genome. This allows for bypassof DNA replication in the target cell, and so enables acceleratedtransgene expression.

The one or more ITRs will preferably flank the polynucleotide sequenceencoding REP1 or a variant thereof at either end. The inclusion of oneor more ITRs is preferred to aid concatamer formation of the vector ofthe invention in the nucleus of a host cell, for example following theconversion of single-stranded vector DNA into double-stranded DNA by theaction of host cell DNA polymerases. The formation of such episomalconcatamers protects the vector construct during the life of the hostcell, thereby allowing for prolonged expression of the transgene invivo.

In preferred embodiments, ITR elements will be the only sequencesretained from the native AAV genome in the derivative. Thus, aderivative will preferably not include the rep and/or cap genes of thenative genome and any other sequences of the native genome. This ispreferred for the reasons described above, and also to reduce thepossibility of integration of the vector into the host cell genome.Additionally, reducing the size of the AAV genome allows for increasedflexibility in incorporating other sequence elements (such as regulatoryelements) within the vector in addition to the transgene.

With reference to the AAV2 genome of SEQ ID NO: 1, the followingportions could therefore be removed in a derivative of the invention:One inverted terminal repeat (ITR) sequence, the replication (rep) andcapsid (cap) genes (NB: the rep gene in the wildtype AAV genome shouldnot to be confused with REP1, the human gene affected in choroideremia).However, in some embodiments, including in vitro embodiments,derivatives may additionally include one or more rep and/or cap genes orother viral sequences of an AAV genome. Naturally occurring AAV virusintegrates with a high frequency at a specific site on human chromosome19, and shows a negligible frequency of random integration, such thatretention of an integrative capacity in the vector may be tolerated in atherapeutic setting.

Where a derivative genome comprises genes encoding capsid proteins i.e.VP1, VP2 and/or VP3, the derivative may be a chimeric, shuffled orcapsid-modified derivative of one or more naturally occurring AAVviruses. In particular, the invention encompasses the provision ofcapsid protein sequences from different serotypes, clades, clones, orisolates of AAV within the same vector i.e. pseudotyping.

Chimeric, shuffled or capsid-modified derivatives will be typicallyselected to provide one or more desired functionalities for the viralvector. Thus, these derivatives may display increased efficiency of genedelivery, decreased immunogenicity (humoral or cellular), an alteredtropism range and/or improved targeting of a particular cell typecompared to an AAV viral vector comprising a naturally occurring AAVgenome, such as that of AAV2. Increased efficiency of gene delivery maybe effected by improved receptor or co-receptor binding at the cellsurface, improved internalisation, improved trafficking within the celland into the nucleus, improved uncoating of the viral particle andimproved conversion of a single-stranded genome to double-stranded form.Increased efficiency may also relate to an altered tropism range ortargeting of a specific cell population, such that the vector dose isnot diluted by administration to tissues where it is not needed.

Chimeric capsid proteins include those generated by recombinationbetween two or more capsid coding sequences of naturally occurring AAVserotypes. This may be performed for example by a marker rescue approachin which non-infectious capsid sequences of one serotype arecotransfected with capsid sequences of a different serotype, anddirected selection is used to select for capsid sequences having desiredproperties. The capsid sequences of the different serotypes can bealtered by homologous recombination within the cell to produce novelchimeric capsid proteins.

Chimeric capsid proteins also include those generated by engineering ofcapsid protein sequences to transfer specific capsid protein domains,surface loops or specific amino acid residues between two or more capsidproteins, for example between two or more capsid proteins of differentserotypes.

Shuffled or chimeric capsid proteins may also be generated by DNAshuffling or by error-prone PCR. Hybrid AAV capsid genes can be createdby randomly fragmenting the sequences of related AAV genes e.g. thoseencoding capsid proteins of multiple different serotypes and thensubsequently reassembling the fragments in a self-priming polymerasereaction, which may also cause crossovers in regions of sequencehomology. A library of hybrid AAV genes created in this way by shufflingthe capsid genes of several serotypes can be screened to identify viralclones having a desired functionality. Similarly, error prone PCR may beused to randomly mutate AAV capsid genes to create a diverse library ofvariants which may then be selected for a desired property.

The sequences of the capsid genes may also be genetically modified tointroduce specific deletions, substitutions or insertions with respectto the native wild-type sequence. In particular, capsid genes may bemodified by the insertion of a sequence of an unrelated protein orpeptide within an open reading frame of a capsid coding sequence, or atthe N- and/or C-terminus of a capsid coding sequence.

The unrelated protein or peptide may advantageously be one which acts asa ligand for a particular cell type, thereby conferring improved bindingto a target cell or improving the specificity of targeting of the vectorto a particular cell population. An example might include the use of RGDpeptide to block uptake in the retinal pigment epithelium and therebyenhance transduction of surrounding retinal tissues (Cronin et al., 2008ARVO Abstract: D1048). The unrelated protein may also be one whichassists purification of the viral particle as part of the productionprocess i.e. an epitope or affinity tag. The site of insertion willtypically be selected so as not to interfere with other functions of theviral particle e.g. internalisation, trafficking of the viral particle.The skilled person can identify suitable sites for insertion based ontheir common general knowledge. Particular sites are disclosed in Choiet al, referenced above.

The invention additionally encompasses the provision of sequences of anAAV genome in a different order and configuration to that of a nativeAAV genome. The invention also encompasses the replacement of one ormore AAV sequences or genes with sequences from another virus or withchimeric genes composed of sequences from more than one virus. Suchchimeric genes may be composed of sequences from two or more relatedviral proteins of different viral species.

The vector of the invention takes the form of a polynucleotide sequencecomprising an AAV genome or derivative thereof and a sequence encodingREP1 or a variant thereof.

For the avoidance of doubt, the invention also provides an AAV viralparticle comprising a vector of the invention. The AAV particles of theinvention include transcapsidated forms wherein an AAV genome orderivative having an ITR of one serotype is packaged in the capsid of adifferent serotype. The AAV particles of the invention also includemosaic forms wherein a mixture of unmodified capsid proteins from two ormore different serotypes makes up the viral envelope. The AAV particlealso includes chemically modified forms bearing ligands adsorbed to thecapsid surface. For example, such ligands may include antibodies fortargeting a particular cell surface receptor.

The invention additionally provides a host cell comprising a vector orAAV viral particle of the invention.

REP1

The vector of the invention further comprises a polynucleotide sequenceencoding a REP1 polypeptide or a variant thereof. The human cDNAsequence for REP1 (or Rab escort protein-1, also known as Rab proteingeranylgeranyltransferase component A) is shown in SEQ ID NO: 2 andencodes the protein shown in SEQ ID NO: 3. A further cDNA sequence forREP1 is shown in SEQ ID NO: 4.

A REP1 polypeptide or variant thereof is any polypeptide which assistsin prenylation of a Rab GTPase protein. The ability of a REP1polypeptide or variant thereof to assist in prenylation of a Rab GTPaseprotein can be routinely determined by a person skilled in the art. Apolynucleotide sequence encoding a variant of REP1 is any sequence whichencodes a protein assisting in prenylation activity for a Rab-1 GTPase.Preferably the sequence encodes a protein which assists in providingsimilar or higher prenylation activity for Rab-1 GTPase compared to thepolypeptide of SEQ ID NO: 3.

More preferably, the polynucleotide sequence encodes SEQ ID NO: 3 or avariant thereof, and is a variant of the polynucleotide sequence of SEQID NO: 2. A variant of SEQ ID NO: 2 or 3 may comprise truncations,mutants or homologues thereof, and any transcript variants thereof whichencode a functional REP polypeptide.

Any homologues mentioned herein are typically at least 70% homologous toa relevant region of SEQ ID NO: 2 or 3. A specific homologue is the REP2polypeptide, which is 75% homologous to REP1, and can functionallycompensate for REP1 deficiency.

Homology can be measured using known methods. For example the UWGCGPackage provides the BESTFIT program which can be used to calculatehomology (for example used on its default settings) (Devereux et al(1984) Nucleic Acids Research 12, 387-395). The PILEUP and BLASTalgorithms can be used to calculate homology or line up sequences(typically on their default settings), for example as described inAltschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990)J Mol Biol 215:403-10. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation (http://www.ncbi.nlm.nih.gov/).

In preferred embodiments, a variant sequence may encode a polypeptidewhich is at least 55%, 65%, 70%, 75%, 80%, 85%, 90% and more preferablyat least 95%, 97% or 99% homologous to a relevant region of SEQ ID NO: 3over at least 20, preferably at least 30, for instance at least 40, 60,100, 200, 300, 400 or more contiguous amino acids, or even over theentire sequence of the variant. The relevant region will be one whichprovides for functional activity of REP1 in assisting in prenylationactivity for a Rab-1 GTPase.

Alternatively, and preferably the variant sequence may encode apolypeptide having at least 70%, 75%, 80%, 85%, 90% and more preferablyat least 95%, 97% or 99% homologous to full-length SEQ ID NO: 3 over itsentire sequence. Typically the variant sequence differs from therelevant region of SEQ ID NO: 3 by at least, or less than, 2, 5, 10, 20,40, 50 or 60 mutations (each of which can be substitutions, insertionsor deletions).

A variant Rep-1 polypeptide may have a percentage identity with aparticular region of SEQ ID NO: 3 which is the same as any of thespecific percentage homology values (i.e. it may have at least 70%, 80%or 90% and more preferably at least 95%, 97% or 99% identity) across anyof the lengths of sequence mentioned above.

Variants of SEQ ID NO: 3 also include truncations. Any truncation may beused so long as the variant is still able to prenylate a Rab-1 GTPasesubstrate polypeptide. Truncations will typically be made to removesequences that are non-essential for prenylation activity and/or do notaffect conformation of the folded protein, in particular folding of theactive site. Appropriate truncations can routinely be identified bysystematic truncation of sequences of varying length from the N- orC-terminus. Preferred truncations are N-terminal and may remove allother sequences except for the catalytic domain.

Variants of SEQ ID NO: 3 further include mutants which have one or more,for example, 2, 3, 4, 5 to 10, 10 to 20, 20 to 40 or more, amino acidinsertions, substitutions or deletions with respect to a particularregion of SEQ ID NO: 3. Deletions and insertions are made preferablyoutside of the catalytic domain as described below. Substitutions arealso typically made in regions that are non-essential for proteaseactivity and/or do not affect conformation of the folded protein.

Substitutions preferably introduce one or more conservative changes,which replace amino acids with other amino acids of similar chemicalstructure, similar chemical properties or similar side-chain volume. Theamino acids introduced may have similar polarity, hydrophilicity,hydrophobicity, basicity, acidity, neutrality or charge to the aminoacids they replace. Alternatively, the conservative change may introduceanother amino acid that is aromatic or aliphatic in the place of apre-existing aromatic or aliphatic amino acid. Conservative amino acidchanges are well known in the art and may be selected in accordance withthe properties of the 20 main amino acids as defined in Table A below.

Similarly, preferred variants of the polynucleotide sequence of SEQ IDNO: 2 include polynucleotides having at least 70%, 75%, 80%, 85%, 90%and more preferably at least 95%, 97% or 99% homologous to a relevantregion of SEQ ID NO: 2. Preferably the variant displays these levels ofhomology to full-length SEQ ID NO: 2 over its entire sequence

TABLE A Chemical properties of amino acids Ala aliphatic, hydrophobic,neutral Met hydrophobic, neutral Cys polar, hydrophobic, neutral Asnpolar, hydrophilic, neutral Asp polar, hydrophilic, charged (−) Prohydrophobic, neutral Glu polar, hydrophilic, charged (−) Gln polar,hydrophilic, neutral Phe aromatic, hydrophobic, neutral Arg polar,hydrophilic, charged (+) Gly aliphatic, neutral Ser polar, hydrophilic,neutral His aromatic, polar, hydrophilic, Thr polar, hydrophilic,neutral charged (+) Ile aliphatic, hydrophobic, neutral Val aliphatic,hydrophobic, neutral Lys polar, hydrophilic, charged (+) Trp aromatic,hydrophobic, neutral Leu aliphatic, hydrophobic, neutral Tyr aromatic,polar, hydrophobic

Promoters and Regulatory Sequences

The vector of the invention also includes elements allowing for theexpression of the REP1 transgene in vitro or in vivo. Thus, the vectortypically comprises a promoter sequence operably linked to thepolynucleotide sequence encoding Rep-1 or a variant thereof.

Any suitable promoter may be used. The promoter sequence may beconstitutively active i.e. operational in any host cell background, oralternatively may be active only in a specific host cell environment,thus allowing for targeted expression of the transgene in a particularcell type. The promoter may show inducible expression in response topresence of another factor, for example a factor present in a host cell.In any event, where the vector is administered for therapy, the promotermust be functional in a retinal cell background.

In some embodiments, it is preferred that the promoter showsretinal-cell specific expression in order to allow for the transgene toonly be expressed in retinal cell populations. Thus, expression from thepromoter may be retinal-cell specific, for example confined only tocells of the neurosensory retina and retinal pigment epithelium.

Preferred promoters for the Rep-1 transgene include the chickenbeta-actin (CBA) promoter, optionally in combination with acytomegalovirus (CME) enhancer element. A particularly preferredpromoter is a hybrid CBA/CAG promoter, for example the promoter used inthe rAVE expression cassette (GeneDetect.com). A further preferredpromoter is shown in SEQ ID NO: 6. Examples of promoters based on humansequences that would induce retina specific gene expression includerhodospin kinase for rods and cones (Allocca et al., 2007, J Virol81:11372-80), PR2.1 for cones only (Mancuso et al. 2009, Nature) and/orRPE65 for the retinal pigment epithelium (Bainbridge et al., 2008, N EngJ Med).

The vector of the invention may also comprise one or more additionalregulatory sequences with may act pre- or post-transcriptionally. Theregulatory sequence may be part of the native REP1 gene locus or may bea heterologous regulatory sequence. The vector of the invention maycomprise portions of the 5′UTR or 3′UTR from the native REP1 transcript.For example, the polynucleotide of SEQ ID NO:4 includes some of the5′UTR sequence from the native REP1 transcript.

Regulatory sequences are any sequences which facilitate expression ofthe transgene i.e. act to increase expression of a transcript, improvenuclear export of mRNA or enhance its stability. Such regulatorysequences include for example enhancer elements, postregulatory elementsand polyadenylation sites. A preferred polyadenylation site is theBovine Growth Hormone poly-A signal which may be as shown in SEQ ID NO:7. In the context of the vector of the invention such regulatorysequences will be cis-acting. However, the invention also encompassesthe use of trans-acting regulatory sequences located on additionalgenetic constructs.

A preferred postregulatory element for use in a vector of the inventionis the woodchuck hepatitis postregulatory element (WPRE) or a variantthereof. The sequence of the WPRE is provided in SEQ ID NO:5. Theinvention encompasses the use of any variant sequence of the WPRE whichincreases expression of the REP1 transgene compared to a vector withouta WPRE. Preferably, variant sequences display at least 70% homology toSEQ ID NO:5 over its entire sequence, more preferably 75%, 80%, 85%, 90%and more preferably at least 95%, 97% or 99% homology to SEQ ID NO: 5over its entire sequence.

Another regulatory sequence which may be used in a vector of the presentinvention is a scaffold-attachment region (SAR). Additional regulatorysequences may be selected by the skilled person on the basis of theircommon general knowledge.

Preparation of Vector

The vector of the invention may be prepared by standard means known inthe art for provision of vectors for gene therapy. Thus, wellestablished public domain transfection, packaging and purificationmethods can be used to prepare a suitable vector preparation.

As discussed above, a vector of the invention may comprise the fullgenome of a naturally occurring AAV virus in addition to apolynucleotide sequence encoding REP1 or a variant thereof. However,commonly a derivatised genome will be used, for instance a derivativewhich has at least one inverted terminal repeat sequence (ITR), butwhich may lack any AAV genes such as rep or cap.

In such embodiments, in order to provide for assembly of the derivatisedgenome into an AAV viral particle, additional genetic constructsproviding AAV and/or helper virus functions will be provided in a hostcell in combination with the derivatised genome. These additionalconstructs will typically contain genes encoding structural AAV capsidproteins i.e. cap, VP1, VP2, VP3, and genes encoding other functionsrequired for the AAV life cycle, such as rep. The selection ofstructural capsid proteins provided on the additional construct willdetermine the serotype of the packaged viral vector.

A particularly preferred packaged viral vector for use in the inventioncomprises a derivatised genome of AAV2 in combination with AAV5 or AAV8capsid proteins. This packaged viral vector typically comprises one ormore AAV2 ITRs optionally as shown in SEQ ID NO: 8 and/or 9, or variantsthereof.

As mentioned above, AAV viruses are replication incompetent and sohelper virus functions, preferably adenovirus helper functions willtypically also be provided on one or more additional constructs to allowfor AAV replication.

All of the above additional constructs may be provided as plasmids orother episomal elements in the host cell, or alternatively one or moreconstructs may be integrated into the genome of the host cell.

In these aspects, the invention provides a method for production of avector of the invention. The method comprises providing a vector whichcomprises an adeno-associated virus (AAV) genome or a derivative thereofand a polynucleotide sequence encoding REP1 or a variant thereof in ahost cell, and providing means for replication and assembly of saidvector into an AAV viral particle. Preferably, the method comprisesproviding a vector comprising a derivative of an AAV genome and apolynucleotide sequence encoding REP1 or a variant thereof, togetherwith one or more additional genetic constructs encoding AAV and/orhelper virus functions. Typically, the derivative of an AAV genomecomprises at least one ITR. Optionally, the method further comprises astep of purifying the assembled viral particles. Additionally, themethod may comprise a step of formulating the viral particles fortherapeutic use.

Methods of Therapy and Medical Uses

As discussed above, the present inventors have surprisingly demonstratedthat a vector of the invention may be used to address the cellulardysfunction underlying choroideremia. In particular, they have shownthat use of the vector can correct the prenylation defect associatedwith choroideremia. This provides a means whereby the degenerativeprocess of the disease can be treated, arrested, palliated or prevented.

The invention therefore provides a method of treating or preventingchoroideremia in a patient in need thereof, comprising administering atherapeutically effective amount of a vector of the invention to thepatient by direct retinal, subretinal or intravitreal injection.Accordingly, choroideremia is thereby treated or prevented in saidpatient.

In a related aspect, the invention provides for use of a vector of theinvention in a method of treating or preventing choroideremia byadministering said vector to a patient by direct retinal, subretinal orintravitreal injection. Additionally, the invention provides the use ofa vector of the invention in the manufacture of a medicament fortreating or preventing choroideremia by direct retinal, subretinal orintravitreal injection.

In all these embodiments, the vector of the invention may beadministered in order to prevent the onset of one or more symptoms ofchoroideremia. The patient may be asymptomatic. The subject may have apredisposition to the disease. The method or use may comprise a step ofidentifying whether or not a subject is at risk of developing, or has,choroideremia. A prophylactically effective amount of the vector isadministered to such a subject. A prophylactically effective amount isan amount which prevents the onset of one or more symptoms of thedisease.

Alternatively, the vector may be administered once the symptoms of thedisease have appeared in a subject i.e. to cure existing symptoms of thedisease. A therapeutically effective amount of the antagonist isadministered to such a subject. A therapeutically effective amount is anamount which is effective to ameliorate one or more symptoms of thedisease. Typically, such an amount increases the level of prenylation ofRab GTPases in the eye. This can be confirmed as described below. Suchan amount may also arrest, slow or reverse some loss of peripheralvision associated with choroideremia. Such an amount may also arrest,slow or reverse onset of nightblindness.

The subject may be male or female. Male subjects show more severesymptoms, since choroideremia is an X-linked disease, but femalesubjects also display symptoms of the disease and occasionally have asevere phenotype. The subject is preferably identified as being at riskof, or having, the disease. The retina may show the characteristicappearance initially of thinning of the choroid and progressing toexposure of the underlying sclera in patches. There may be loss ofamplitude of the electroretinogram peripherally. In many cases there maybe a family history of choroideremia. Usually, but not always, amutation may be identified in the REP1 gene located on the X-chromosome.

The administration of the vector is typically by direct retinal orsubretinal injection. This includes direct delivery to cells of theneurosensory retina and retinal pigment epithelium, such as epithelialor photoreceptor cells. The delivery is made typically directly to orsubretinally to the degenerating retina in a choroideremia patient. Thevector may transduce the above target cells without entering any othercell populations. Intravitreal injection may also be used to deliver thevector of the invention. The delivery may not be subretinal or may notbe by subretinal injection. The delivery may not be transvitreal.

The dose of a vector of the invention may be determined according tovarious parameters, especially according to the age, weight andcondition of the patient to be treated; the route of administration; andthe required regimen. Again, a physician will be able to determine therequired route of administration and dosage for any particular patient.

A typical single dose is between 10¹⁰ and 10¹² genome particles,depending on the amount of remaining retinal tissue that requirestransduction. A genome particle is defined herein as an AAV capsid thatcontains a single stranded DNA molecule that can be quantified with asequence specific method (such as real-time PCR). That dose may beprovided as a single dose, but may be repeated for the fellow eye or incases where vector may not have targeted the correct region of retinafor whatever reason (such as surgical complication). The treatment ispreferably a single permanent treatment for each eye, but repeatinjections, for example in future years and/or with different AAVserotypes may be considered.

The invention also provides a method of monitoring treatment orprevention of choroideremia in a patient comprising measuringprenylation activity ex vivo in retinal cells obtained from said patientfollowing administration of the AAV vector of the invention by directretinal, subretinal or intravitreal injection. This method allows fordetermination of the efficacy of treatment.

Pharmaceutical Compositions

The vector of the invention can be formulated into pharmaceuticalcompositions. These compositions may comprise, in addition to thevector, a pharmaceutically acceptable excipient, carrier, buffer,stabiliser or other materials well known to those skilled in the art.Such materials should be non-toxic and should not interfere with theefficacy of the active ingredient. The precise nature of the carrier orother material may be determined by the skilled person according to theroute of administration, i.e. here direct retinal, subretinal orintravitreal injection.

The pharmaceutical composition is typically in liquid form. Liquidpharmaceutical compositions generally include a liquid carrier such aswater, petroleum, animal or vegetable oils, mineral oil or syntheticoil. Physiological saline solution, magnesium chloride, dextrose orother saccharide solution or glycols such as ethylene glycol, propyleneglycol or polyethylene glycol may be included. In some cases, asurfactant, such as pluronic acid (PF68) 0.001% may be used.

For injection at the site of affliction, the active ingredient will bein the form of an aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

For delayed release, the vector may be included in a pharmaceuticalcomposition which is formulated for slow release, such as inmicrocapsules formed from biocompatible polymers or in liposomal carriersystems according to methods known in the art.

EXAMPLES

The present Examples describe a model for testing therapeutic strategiesfor choroideremia and correction of the disease phenotype. Geneticconstructs, consisting of a promoter, the REP1 cDNA, and 3′ regulatoryelements, when packaged into a recombinant viral vector, are shown toefficiently transduce target cells within the degenerating retina.

Example 1

Cloning of Human REP1 cDNA, and Generation of the CBA-REP-1-WPREExpression Cassette, Construction of pAAV-CBA-REP-1-WPRE-bGHpA andPackaging of AAV REP-1 Virus.

A cDNA of human REP1 was isolated from a human cDNA library using PCRamplification and primers homologous to the known REP1 sequence The cDNAisolated was sequenced and shown to be homologous to the knowntranslated Variant 1 of the REP1 mRNA sequence as deposited intoGenbank, Accession Number NM_000390. The cDNA has the sequence of SEQ IDNO: 4.

This cDNA was inserted into a pAAV cis plasmid, termed pAM. pAM is ahigh copy number plasmid originally derived from pBR322, but includesstabilized AAV-2 left and right inverted terminal repeats which flankthe expression cassette of choice. For the AAV-REP1 vector, a modifiedCBA/CAG promoter (chicken beta-actin with CMV enhancer) was used todrive expression of REP1 and a modified WPRE sequence and bGH polyA wereprovided 3′ to the cDNA. This plasmid was termedpAAV2-CBA-hREP-1-WPRE-bGH, (pAAV-REP-1).

pAAV-REP-1 was used to generate recombinant AAV-Rep-1 using wellestablished and public domain triple transfection packaging andpurification methods Vector stocks generated using this method varied ingenomic titer, but most commonly the stocks obtained followingpurification were 10¹²-10¹³ gp/ml (gp=genome particles—see above). Thestocks were subsequently diluted for in vivo use as described below.

Example 2

Expression of REP1 from Vector in Human Choroideraemia (Chm) Cells

Expression of REP1 from the AAV2 REP1 vector was evaluated in humanchoroideremia (Chm) fibroblasts. These fibroblast cells were obtainedwith ethical consent from a skin biopsy taken from a choroideraemiapatient. Expression of GFP from a control vector served as a control. Asa prelude to the work with human cells, expression was also confirmedafter subretinal injection of the AAV.REP1 vector in mice by Westernblot, as the antibody probe recognises the human but not mouse forms ofREP1 protein.

Results are shown in FIG. 1. REP1 was not detected by immunoblottingwith an anti-hREP1 antibody in nontransduced Chm fibroblasts (lane 2),whereas REP1 is detected in normal (WT=human wildtype) fibroblasts (lane1). Following transduction with the AAV2.REP1 vector, at equal doses of40 μg of lysate and dilution to 5 μg it can be seen that the level ofhREP1 expressed by the AAV2.REP1 vector in Chm cells is approximatelyten fold higher (lanes 3-6) than the levels in wildtype cells (lane 1).No toxic effects on cell growth were observed with this degree ofover-expression.

Example 3

Correction of Prenylation Defect by Vector in Chm Cells

Choroideraemia mice do not have a retinal degeneration phenotype in thesame way as human patients so it is not possible to perform a directassessment of retinal rescue using a gene therapy approach. For thisreason, correction of the disease phenotype was assessed in human Chmcells in vitro.

Results are shown in FIG. 2. Transduction with the AAV2.REP1 showed acorrection of the prenylation defect seen in Chm cells, raising theprenylation activity to significantly higher than normal levels aftertreatment of 2×10⁵ cells with 1.5×10¹⁰ viral genome particles ofAAV2.REP1. This confirms that the AAV2.REP1 vector expresses functionalREP1 protein in human cells affected by choroideraemia.

In more detail, the normal prenylation activity in wildtype (WT)fibroblasts yields approximately 0.32 pmol of [3H]-GGPP; inchoroideremia (Chm) fibroblasts this is reduced to 0.19 pmol. Asexpected the prenylation activity was unchanged following transductionof the Chm fibroblasts with the AAV.GFP control vector. Followingtransduction with the AAV2.REP1 vector, however, the prenylationactivity increased significantly to yield 0.42 pmol of [3H]-GGPP (n=4,p<0.01).

Example 4

Targeted In Vivo Expression of Reporter Gene from Vector in Mice

To confirm the ubiquitous activity of the CBA promoter and regulatorysequences in the AAV2 vector, the gene encoding REP1 was replaced with areporter gene encoding green fluorescent protein (GFP) to createAAV2.CBA.GFP.WPRE.BGH (AAV2.GFP). GFP was selected to evaluate in vivoexpression, since although easy to identify on Western blots, the humanREP1 protein is not easily detected by indirect immunohistochemistry onretinal sections.

The AAV2.GFP construct was injected into the mouse subretinal space andexpression of GFP was monitored by microscopy. Results are shown in FIG.3, which confirm that the vector had the predicted tropism for both theneurosensory retina and retinal pigment epithelium. This confirms thecapsid sequence and regulatory elements lead to high levels of geneexpression in photoreceptors and the retinal pigment epithelium.

Example 5

Toxicity Study

Doses of AAV2.REP1 vector were injected into the subretinal space ofwild-type mice (n=9) in order to determine any possible toxic effects inretinal function at the very highest doses. We tested vectorconcentrations in mice (1×10¹¹ and 1×10¹² gp per ml) that were a logunit higher than proposed high and low concentrations to be used inpatients (10¹° and 10″ gp per ml).

Results are shown in FIG. 4. No toxic effects on the electroretinogram(ERG) were detected six months after subretinal injection with eitherthe high (n=5) or low (n=4) dose of AAV.REP1 vector. To control for anynon-specific effects of retinal surgery or the AAV2 vector, the felloweye had a very similar subretinal injection and titre of AAV2.GFP.

At the highest AAV2.GFP dose there was a mild reduction in the ERGamplitude, which reflects a mild known toxic effect using the maximaldose of vector expressing GFP with this strong promoter, and confirmsthe sensitivity of this test in detecting a dose-related effect.Nevertheless there was no detectable ERG reduction in AAV2.REP1 treatedeyes at either dose which suggested that REP1 over-expression in theretina was less toxic than GFP.

1. A vector, which comprises an adeno-associated virus (AAV) genome or aderivative thereof and a polynucleotide sequence encoding REP1 or avariant thereof.
 2. A vector according to claim 1, wherein saidderivative is a chimeric, shuffled or capsid modified derivative.
 3. Avector according to claim 1 or 2, wherein said AAV genome is from anaturally derived serotype or isolate or clade of AAV.
 4. A vectoraccording to claim 3, wherein said serotype is AAV serotype 2 (AAV2). 5.A vector according to claim 4, which comprises SEQ ID NO: 1 or aderivative thereof.
 6. A vector according to any one of the precedingclaims, wherein said polynucleotide sequence encodes a polypeptidehaving at least 70% homology to SEQ ID NO: 3 over its entire sequence.7. A vector according to claim 6, wherein said polynucleotide sequencehas at least 70% homology to SEQ ID NO: 2 over its entire sequence.
 8. Avector according to any one of the preceding claims, which comprises apromoter sequence operably linked to said polynucleotide sequenceencoding REP1 or a variant thereof.
 9. A vector according to claim 8,wherein said promoter is constitutively active.
 10. A vector accordingto claim 8, wherein expression from said promoter is retinal-cellspecific.
 11. A vector according to any one of the preceding claims,which comprises one or more additional regulatory sequences.
 12. Avector according to claim 11, which comprises a sequence having at least70% homology to SEQ ID NO: 5 over its entire sequence.
 13. A method oftreating or preventing choroideremia in a patient in need thereof,comprising administering a therapeutically effective amount of a vectoraccording to any one of the preceding claims to said patient by directretinal, subretinal or intravitreal injection, and thereby treating orpreventing choroideremia in said patient.
 14. A method according toclaim 13, wherein said vector is administered directly into thesubretinal space.
 15. A vector according to any one of claims 1 to 12for use in a method of treating or preventing choroideremia byadministering said vector to a patient by direct retinal, subretinal orintravitreal injection.